The invention relates to a method of determining incompatibility-reaction-causing substances in therapeutically and prophylactically applicable blood products as well as use of this method for testing the safety of these products.
The invention further relates to a method of inactivating incompatibility-reaction-causing substances in therapeutically or prophylactically applicable immunoglobulin-containing blood fractions while using proteolytically active enzymes. In particular, the invention relates to a method of inactivating such substances at the production of new immunoglobulin-G-containing fractions from human or animal plasma suited for intravenous application.
Immunoglobulin-containing preparations may be applied in case of primary and secondary immune defects, A-gammaglobulinemia or hypogammaglobulinemia, antibody deficiency syndrome, virus infections or bacterial infections.
For obtaining immunoglobulin-containing preparations from human or animal plasma, various methods are already known, e.g. the precipitation with ethanol (J. L. Oncley, M. Melin, D. A. Richart, J. W. Cameron and P. M. Gross, J. Am. Chem. Soc. 71, 541 (1949)) as well as modified ethanol methods according to H. F. Deutsch, L. F. Gosting, R. A. Alberty and J. W. Williams, J. Biol. Chem. 164, 109 (1946) as well as P. Kistler and H. Nitschmann, Vox Sanguinis 7, 414 (1962).
Furthermore, a method is known according to which immunoglobulin is precipitated from plasma by means of ammonium sulfate and polyethyleneglycol (A. Polson, G. M. Potgieter, J. F. Largrier, G. E. F. Mears and F. J. Jourbet, Biochim. Biophys. Acta. 82, 463 (1964)). According to other methods, the use of ion exchangers has been suggested (E. A. Peterson and H. A. Sober, J. Am. Chem. Soc. 78, 751 (1956)).
These methods had the disadvantage that the obtained preparations were suited for intramuscular application only. With intravenous application, they exhibited undesired side reactions, such as vasoactive effects.
Therefore, efforts have been made to reduce side reactions or side effects, to which end immunoglobulin-containing preparations were treated with soluble proteolytic enzymes, such as pepsin, plasmin, papain and others (German Patent No. 1,148,037, as well as Swiss Patent No. 392,780). However, by this treatment the molecular structure of the immunoglobulins is changed, which may result in a shortened biologic half-life period or in undesired side effects, such as vasoactive or leucopenic activity. It was also found out that enzyme residues remain in the preparations, thereby contaminating the same. The storability is accordingly low, the danger of a progressing proteolytic cleavage is great.
German Offenlegungsschrift No. 28 46 412 describes a method of producing immunoglobulins immunologically modified in their Fc-portion, wherein the immunoglobulin-containing fraction is treated with a protease solution. These products are said to have an antiallergic effect in local or systemic application. The method is unsuited for the production of an intravenously applicable preparation, as the protease solution is not or not completely removable from the product and continues with its decomposing effect.
In German Offenlegungsschrift No. 29 36 047 a method for the production of an intravenously administrable immunoglobulin preparation is described, in which a combined purification with ammonium sulfate and polyethyleneglycol is carried out in the presence of a soluble carbohydrate or of a polyol. It is true that vasoactive side effects have been eliminated, yet an improvement in terms of safety and reproducibility in case of intravenous application is still desirable.
Among the prior art, also the Japanese patent applications published under Nos. 56-7721 and 56-15215 as well as German Offenlegungsschrift No. 32 20 309 are to be mentioned, which have as their objects methods for the production of intravenously applicable immunoglobulin preparations. The treatment is to be effected with immobilized plasmin or immobilized pepsin, yet the results attainable thereby are not satisfactory, either, because the preparations have an undesirably high anticomplementary activity.